1. Technical Field
This invention relates to the measurement of dissolved oxygen concentrations in solution.
2. Description of the Background Art
Fluorescent probes for oxygen sensing are of great interest because of their high sensitivity and potential specificity. Importantly, metal ligand complexes display luminescent decay times from 100 ns to 100 .mu.s. As a consequence, metal ligand complex probes extend the observable time scale of decay measurements by orders of magnitude over other routinely used fluorophores. A variety of fluorophores with longer lifetimes have been used as indicators of dissolved oxygen (Vaughan, W. M. et al., Biochemistry 9:464-473 (1970); Cox, M. E. et al., App. Optics 24(14):2114-2120 (1985)).
Quenching of fluorescence by oxygen is one of the earliest observations in fluorescence quenching. Long lifetime phosphorescent porphyrins also have been developed for this use (Papkovsky, D. B. et al., Anal. Chem., 67:4112-4117 (1995)). One of the most widely used oxygen sensors has been ruthenium-(4,7-diphenyl-1,10-phenanthroline).sub.3 ([Ru(dpp).sub.3 ].sup.2+). The favorable optical properties of this complex have resulted in considerable effort to improve the performance of oxygen sensors based on this luminescent metal-ligand complex. Additionally, sensors based on [Ru(dpp).sub.3 ].sup.2+ are highly stable and can be steam sterilized (Bambot, S. B. et al. Biotech. Bioengr., 43:1139-1145 (1994)), facilitating use for medical purposes.
To further improve the spectral properties of these long lifetime oxygen sensors, metal-ligand complexes excitable with green light have been developed, as have oxygen sensors which can be excited above 600 nm. These long wavelength oxygen sensors allow measurements through skin (Bambot, S. B. et al., Biosensors & Bioelectronics 10:643-652 (1995)), allowing minimally invasive transdermal sensing of oxygen concentration in body tissues.
The measurement of decay times of the oxygen sensor instead of its fluorescence intensity is preferred in this minimally invasive oxygen sensing in the body because decay times can be easily measured in turbid media and through skin (Szmacinski, H. et al., Sensors and Actuators B 30:207-215 (1996)).
However, all the previously known fluorophores were insoluble in water and were either dissolved in organic solvents, or were contained in polymeric or silicon supports (Bacon, J. R. et al., Anal. Chem., 59:2780-2785 (1987)). These fluorophores thus were not desirable for use in the body of a living animal or for samples incompatible with organic solvents.
In the past, tissue hypoxia was diagnosed in critically ill patients by an indirect method which was time-intensive and required simultaneous measurements of arterial and venous hemoglobin saturation and measurements of cardiac output and lactate concentration. This prior art method was difficult to perform, and revealed little about the oxygen concentration in tissues or in any particular tissues of interest. Therefore, there is a need for improved methods and oxygen sensors, and which can allow immediate determination of oxygen concentrations in the tissues of the body without the need for techniques such as phlebotomy.